Live dead blue stain

For γδ T cell proliferation, cells were labeled with 2.5 μM CFSE (Molecular Probes) at 37°C for 7 min. Cells were seeded in a 96-well plate pre-coated with anti-CD3ε and anti-CD28. After incubation at 37°C for 48h, cells were stained with Live/Dead Blue Stain (Invitrogen) before being analyzed by flow cytometry for CFSE dilution. When indicated 15 U ml^-1 rIL-2 (R&D Systems) or 10 mM L-NMMA (Calbiochem), 0.5 mM N6-(1-iminoethyl)-l-lysine dihydrochloride (L-NIL, Calbiochem) were added to the cultures.
For metabolic assays, γδ T cells were sorted from WT and Nos2KO mice and cultured for 4 days in 0.1 μg ml^-1 anti-CD3ε, 10 μg ml^-1 anti-CD28, 15 μg ml^-1 rIL-7 and 15 U ml^-1 rIL-2. Metabolism analyses were performed directly or after 18h of resting followed by 4h of stimulation in media containing 5 mM L-NMMA and or 15 U ml^-1 rIL-2 when indicated. 5.10⁵ cells per well were plated in Seahorse plates coated with CellTak (Corning). The OCR and ECAR were measured in XF medium (unbuffered RPMI containing 2 mM glutamine, pH 7.4) under basal conditions and in response to glucose (25 mM), oligomycin (1 μM), FCCP (1.5 M) plus pyruvate (1 mM) and antimycin A (1 μM) plus rotenone (0.1 μM) with an XF-24 Extracellular Flux Analyzer (Seahorse Bioscience). Rate of glycolysis was defined as the difference between ECAR following the injection of glucose and the basal ECAR reading.

Surface staining was performed by incubating cells on ice, for 20 min, with saturating concentrations of labeled Abs in PBS, 5% FCS and 0.5% EDTA. Mouse cell-staining reactions were preceded by a 15-min incubation with purified anti-CD16/32 Abs (FcγRII/III block; 2.4G2) obtained from hybridoma supernatants. Intracellular cytokine staining were performed after stimulation of single cell suspensions with Phorbol 12-myristate 13-acetate (PMA) (50 ng ml^-1) (Sigma), ionomycin (0.5 μg ml^-1) (Sigma) and 1 μL ml^-1 Golgi Plug (BD Biosciences) for 4h at 37°C 5% CO₂. Cells were incubated with Live/Dead Blue stain (Invitrogen), according to the manufacturer protocol prior to Ab surface staining. Then, intracellular staining was performed using Cytofix/Cytoperm kit (BD biosciences) following the manufacturer’s instructions. γδ T cell apoptosis were assessed ex vivo by staining pLNs for FITC-conjugated annexin V (BioLegend) according to the manufacturer’s instructions. Data files were acquired and analyzed on LSRII using Diva software (BD Biosciences).